Phylogenetic and genetic characterization of influenza A H9N2 viruses isolated from backyard poultry in selected farms in Ghana

Abstract Introduction Avian influenza viruses (AIV) cause significant economic losses to poultry farmers worldwide. These viruses have the ability to spread rapidly, infect entire poultry flocks, and can pose a threat to human health. The National Influenza Centre (NIC) at the Noguchi Memorial Institute for Medical Research in collaboration with the Ghana Armed forces (GAF) and the U.S. Naval Medical Research Unit No. 3, Ghana Detachment (NAMRU‐3) performs biannual surveillance for influenza viruses among poultry at military barracks throughout Ghana. This study presents poultry surveillance data from the years 2017 to 2019. Methodology Tracheal and cloacal swabs from sick and healthy poultry were collected from the backyards of GAF personnel living quarters and transported at 4°C to the NIC. Viral ribonucleic acid (RNA) was isolated and analyzed for the presence of influenza viruses using real‐time polymerase chain reaction (PCR) assays. Viral nucleic acids extracted from influenza A‐positive specimens were sequenced using universal influenza A‐specific primers. Results Influenza A H9N2 virus was detected in 11 avian species out of 2000 samples tested. Phylogenetic analysis of viral haemagglutinin (HA) protein confirms the possibility of importation of viruses from North Africa and Burkina Faso. Although the detected viruses possess molecular markers of virulence and mammalian host adaptation, the HA cleavage site anlaysis confirmed low pathogenicity of the viruses. Conclusions These findings confirm the ongoing spread of H9 viruses among poultry in Ghana. Poultry farmers need to be vigilant for sick birds and take the appropriate public health steps to limit the spread to other animals and spillover to humans.


INTRODUCTION
Influenza is a disease of global public health significance; causing 3-5 million severe illnesses each year resulting in up to 650,000 respiratory deaths in annual seasonal epidemics (Paget et al., 2019). Influenza was reported in Ghana as early as 1918 when the Spanish flu pandemic led to the death of some 100,000 people in a population of 4 million in the then Gold Coast and is described as the worst short-term demographic disaster in the known history of Ghana (Patterson, 1983). In addition to the global health effects of influenza on international public health systems in Ghana, influenza also remains a high priority respiratory pathogen of U.S. military relevance. Respiratory surveillance activities such as these are essential to better understand the transmission dynamics, epidemiologic trends and emergence of new strains or circulating variants to guide military force health protection (FHP) measures.
Influenza viruses are enveloped and belong to the viral family Orthomyxoviridae which are characterized by segmented, singlestranded, negative sense RNA genomes (Knipe & Howley, 2007). The family consists of seven different genera: influenza viruses A, B, C and D; Isaviruses; Thogotoviruses; and newly proposed Quarjaviruses (made up of Quaranfil, Johnston Atoll and Lake Chad viruses) (Hause et al., 2014;Knipe & Howley, 2007;Presti et al., 2009). Influenza A viruses (IAV) are responsible for seasonal infections (Knipe & Howley, 2007) as well as occasional pandemics in the human population, leading to elevated mortality rates. IAVs are known to infect humans as well as a wide range of animals including pigs, birds, cats, dogs, seals and horses (Horimoto & Kawaoka, 2005;Knipe & Howley, 2007;Mänz et al., 2013). With the exception of H17-18 and N10-11, all influenza subtypes have been isolated from wild aquatic birds, which are therefore considered the natural reservoirs of IAVs (Knipe & Howley, 2007).
Among the IAV are avian influenza viruses (AIV) such as those belonging to the haemagglutinin (HA) type 5, 7 and 9 groups: these are typically of immense public health concern as they continue to cause significant economic losses to poultry farmers worldwide, due to their ability to spread quickly and infect entire flocks of poultry. AIVs are also a threat to human health including both civilian and millitary populations (Peiris et al., 2007).
In Ghana, troops returning from missions abroad import new breeds of birds that have been identified to be a major source of avian pathogens, including but not limited to AIV. Therefore, The National

Methodology
Biannual surveillance for AIV included sampling of poultry in all poultry-keeping units in military barracks across the country. Seemingly healthy bird populations were randomly sampled, whereas morbid birds (such as those presenting with ruffled feathers, cyanosis, watery diarrhoea) were all sampled when encountered. Tracheal and/or cloacal swabs specimens were collected from sampled birds, packaged and shipped on cold-chain to the NIC for influenza testing.
At the NIC, ribonucleic acid (RNA) was extracted from each sample using the QIAamp viral RNA mini kit. Screening for the presence of IAV was conducted and IAV-positive samples were subsequently subtyped. Master-mix preparations were performed using the AgPATH (ID) One-step RT-PCR kit, with primers and probes, and protocols accessed either from the International Reagents and Resources or from the WHO (WHO, 2017). All PCR testings were performed on the ABI 7500 real-time PCR device. All gene fragments of the subtype AH9N2 viruses detected were amplified using the universal IAVamplifying primers described by Zhou et al. (2009). Briefly, the PCR amplicons, after confirmed on a 1% agarose gel, was quantitated and normalized to about 100 ng using the Qubit fluorimeter. Normalized amplicons were tagmented (fragmented and tagged) using a bead-link transposome. Tagged nucleic acid fragments were cleaned up by trapping beads with a magnetic stand and performing wash cycles. Subsequently, tagged nucleic acids were PCR amplified using primers linked to index adapters to prepare libraries. Libraries were cleaned up, and aliquots of the libraries were pooled, quantified and set up for sequencing on the Miseq using 2 × 151 base pair paired end sequencing. were further assembled to generate a consensus sequence for each HA. Consensus sequences were viewed using the BioEdit software v7.2.5, and a nucleotide BLAST on the NCBI platform using these sequences returned aligning sequences, of which the highest identities (> 90%) were downloaded for further alignment with all reference clade-specific HA sequences. Consensus sequences, BLAST returned sequences and other A H9 sub clades (G1-like, Y280-like and Koreanlike viruses) were used to generate a maximum-likelihood phylogenetic tree applying 1000 bootstrap replicates.

RESULTS
Of 2000 birds tested, 11 (comprising five sick and six healthy birds) were confirmed from five of nine sites in five regions sampled by both PCR and sequencing, as the H9N2 subtype of AIV. A map of Ghana highlighting the areas where H9N2 viruses were identified is shown in Figure 1, while details of identified H9N2 viruses and animal source are shown in Table 1 (Tables 1 and 2) showed that 10/11 viruses possessed the RSSR*GLF cleavage motif on the HA, which is a characteristic of low pathogenic AIV (Nagarajan et al., 2009). One of the 11 viruses had an HA segment that could not be sequenced due to a relatively higher threshold cycle (Ct) value (i.e., Ct > 29).
Multiple markers of mammalian adaptivity, virulence and humanto-human transmission were identified in the HA, matrix, polymerase basic 1 (PB1), and polymerase basic 2 (PB2) proteins as well as in the polymerase acidic (PA) segment and the nucleoprotein (NP) ( Table 2).

DISCUSSION
Phylogenetic analysis showed that the identified viruses clustered closely with viruses detected earlier in North Africa, supporting the specualtion that these viruses were imported into Ghana from North  Pu et al. (2015), this finding forms a basis that these H9 viruses, although of low pathogenicity, could be gradually disseminating throughout Ghana.
As shown by the genetic analyses, observed single nucleotide polymorphisms associated with mammalian adaptation, virulence and/or enhanced zoonotic transmission are of utmost concern for the likeli-

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
This epidemiologic activity was approved by the Noguchi Memorial

DATA AVAILABILITY STATEMENT
Data is available from corresponding author upon reasonable request.